The present invention is directed generally to automated or semi-automated liquid assay apparatus for conducting assays of various liquids, especially biological fluids, for substances contained therein. More specifically, the principles of this invention can be used especially advantageously as an improvement to apparatus such as the Abbott TDx.RTM. Analyzer, to more advantageously perform diagnostic assays of such biological fluids, e.g., immunoassays and nephelometric assays.
The TDx.RTM. Analyzer, commercially available from Abbott Laboratories, Abbott Park, Ill., is a well-known, automated instrument generally used to measure analyte concentrations in biological fluids such as serum, plasma and whole blood. The TDx.RTM. system is designed for use by trained clinical laboratory technicians in hospital laboratories, in private laboratories and in physicians' offices.
The TDx.RTM. Analyzer was originally designed to operate utilizing fluorescence polarization techniques. Fluorescence polarization techniques are based on the principle that a fluorescent labeled compound in solution, when excited by plane polarized light, will emit fluorescence having a degree of polarization related to its molecular rotational relaxation time. The molecular rotational relaxation time, and hence the magnitude of the fluorescence polarization response, is directly related to the molecular size of the compound. Accordingly, when plane polarized light is passed through a solution containing a relatively high molecular weight fluorescent compound, the degree of polarization of the emitted light will in general be greater than when plane polarized light is passed through a solution containing a low molecular weight fluorescent compound.
The fluorescence polarization principle is utilized in the TDx.RTM. Analyzer by mixing a sample containing an analyte (or suspected of containing an analyte) with a "tracer,", i.e., a labelled compound similar to the analyte but capable of producing a fluorescence polarization response to plane polarized light. The analyte is generally a low molecular weight compound. Antibody specific to the analyte and the tracer is also included in the mixture. The tracer and the analyte compete for a limited number of receptor binding sites on the antibody. The amount of tracer that will bind is inversely related to the concentration of analyte in the sample, because the analyte and tracer each bind to the antibody in proportion to their respective concentrations.
The fluorescence polarization response of the solution to plane polarized light will give a quantitative indication of the relative amount of free and bound tracer, because of the discrepancy in molecular size between the former and the latter. The free tracer (i.e., the tracer in solution when not complexed to the antibody) is generally a relatively small molecule compared to the tracer-antibody complex, and will tend to exhibit a shorter rotational relaxation time, such that the incident plane polarized light becomes depolarized. In contrast, plane polarized light interacting with bound tracer will tend to remain highly polarized because the large antibody-tracer rotates very little between the time that light is absorbed and emitted.
Fluorescence polarization techniques usually are applied to analytes of relatively low molecular weight. Since the tracer employed must generally resemble the analyte in order to compete effectively for antibody receptor sites, the tracer itself, in such instances, will be relatively large and will tend to retain the polarization of plane polarized light. Accordingly, when this large tracer molecule is bound to the antibody, there will generally not be an appreciable difference in the fluorescence polarization response when compared with the response produced by the free tracer, so in such cases it may be necessary to consider alterative assay techniques, such as nephelometry.
Nephelometric techniques have been found to provide a means for measuring the light scattered from a solution containing large molecules or suspended particles. In accordance with these techniques, incident light is passed through a solution, a portion of the incident light is scattered, and then the amount of scattered light is measured. These techniques have application, for example, when immunoprecipitation assays are conducted. In such assays, antibodies are raised to the analyte, often forming large three-dimensional lattices. These lattices produce an increase in the light scattering properties of the solution.
The TDx.RTM. Analyzer, for example, provides capabilities for both fluorescence polarization and nephelometric analysis, as well as for other systems of analysis. Whatever assay system is employed, in this Analyzer, as in various other instruments of the prior art, the assays have heretofore been carried out by well-known techniques which involve dispensing reagents from bulk containers located remotely from the test samples undergoing analysis, and mixing the reagents with the samples in reaction cuvettes while the latter are indexed, by means of a carrier comprising a rotating carousel or the like, from one analysis station to another. Although this methodology has proved to be generally satisfactory, it would be economical and useful, in terms of ease of analysis and speed, and advantageous in terms of reagent stability during storage prior to performance of the assays, to provide modification of the reagent and sample containment portions of such instruments which would enable substantially all of the reagents initially to be present in a single, sealed, self-contained device, in amounts which are appropriate for performing a singular, particular assay, and in close proximity to the test sample for ease of dispensing, rather than such reagents being dispensed by the instrument from bulk containers remotely located from the carrier for the samples. Accordingly, there exists a need for such modification and devices and assay methods which utilize such modifications, which would improve the performance of assays using such readily available analytical instrumentation.